Organotypic Culture of Cerebellar Slice for Studies of Developmental Neurotoxicity of MethylmercuryWilliam D. Atchison, PhDMichigan State UniversityMethylmercury (MeHg) is extremely toxic and among the most common forms of mercury found in the environment. MeHg causes prominent neurotoxicicity. The developing brain is especially sensitive to MeHg. Our goal is to understand the mechanisms responsible for this selective vulnerability of certain cells in the brain to MeHg. During development, neurons migrate from one region of the brain to another. This process, can be studied using cultures of brain slices of neonatal rat. Migration of these neurons involves the complex interactions between chemical messengers known as neurotransmitters which either excite, or inhibit electrical activity in the neurons. We propose to develop this system, in our lab for studies of developmental neurotoxicity with MeHg. This preparation can be used for studies of acute and semichronic exposure to MeHg, thus reducing the need for semichronic injections of neonatal animals with MeHg. Furthermore, more than one preparation can be made from a single rat brain, animal, again reducing the overall number of animals needed for a series of studies. Because this preparation maintains the normal intact and developing synaptic connections, it offers a greater advantage over cell culture systems in which the normal connections between neurons (known as circuits) are lost. Movement of cerebellar neurons (in particular, a group called the "granule cells") and concurrent measures of changes in calcium within the cell will be studied using confocal laser scanning microscopic measures in response to acute and semichronic exposure to MeHg. These studies will be an important contribution to the understanding of developmental neurotoxicity with MeHg.Non-animal methodology to assess developmental toxicologyKathleen M. Brundage, PhDWest Virginia UniversityIn the body, the immune system is important for maintaining the health of an individual by detecting and destroying damaged cells, bacteria and viruses. An important player in this system is the white blood cell known as a B lymphocyte or B cell. B cells are responsible for making proteins known as antibodies that specifically attach themselves to bacteria or viruses aiding in their elimination from the body. Little is known about the consequences of chemical exposure on the developing human immune system (immunotoxicity). The majority of studies that have been performed have used animal data to assess human risk. In this study we will use human umbilical cord blood cells to assay the effect that chemicals have on B cell development. Ethanol (alcohol) will be used to test this assay system. Ethanol was chosen for several reasons including that fetal exposure to ethanol is common due to consumption of alcohol by pregnant women and mouse studies have demonstrated a decrease in the number of certain types of B cells in mice exposed in the womb to ethanol. A second part of this project will be to identify protein markers that may be useful in developing screening methods for immunotoxicity. The overall goal of this project is to establish a safe, reliable, non-animal method for evaluating immunotoxicity of B cells in the human immune system.An in vitro flow adaptation chamber replaces animals in an ischemia/reperfusion model to study oxidant-induced signalingShampa Chatterjee, PhDUniversity of PennsylvaniaIschemia reperfusion (I/R), the temporary stop of blood supply to an organ followed by its reinstatement, is a cause of severe injury following surgery, organ transplantation or other kinds of obstruction. This injury is caused by an entity called the reactive oxygen species (ROS) generated by the lining of the blood vessels. In addition to injury, ROS also play a part in altering regular cellular processes and cause tissue and organ dysfunction. A better understanding of these processes would help in reduction of injury following I/R; however such studies also necessitate an extensive and indiscriminate use of animals. Here, we propose to drastically reduce the use of animals by an artificial capillary system (Fibercell) consisting of hollow fibers that can be coated with cells kept under flow using a pump so as to mimic the inner lining of blood vessels. I/R can be performed by stopping and/or restarting the flow in the system. The cells used to line the fibers are isolated from the blood vessels of a few mice and rats. These cells are then expanded into larger cultures thereby preventing large scale sacrifice of animals. Earlier studies from our group have shown that ROS generation with I/R is triggered by the closure of a potassium channel on the cell surface. Therefore potassium channel openers will be used during I/R and ROS generation and the consequent cell damage will be examined to evaluate the potential therapeutic applications of potassium channel openers.Development of a High Throughput Method to Screen for Gene Expression Changes for the Prediction of the Skin Sensitization Potential of ChemicalsG. Frank Gerberick, PhDThe Procter & Gamble CompanyAllergic contact dermatitis is a frequent occupational health problem and is the most common type of chemical allergy in humans. Therefore, there is a need to identify those chemicals that can cause skin sensitization. For this purpose guinea pig, and more recently mouse test methods have been used. However, there is a need to eliminate completely the use of animals in skin sensitization safety assessments. In recent years, our understanding of the cellular mechanisms involved in allergic contact dermatitis has increased substantially. It is known that dendritic cells (DC) residing in the skin play a key role in the development of allergic contact dermatitis. Given the importance of these cells in the initiation of skin sensitization, it seems appropriate to explore whether there are opportunities to develop alternative approaches to hazard identification based upon chemical-induced changes in the gene expression profile of these cells. This project will explore the use of Luminex® xMAP® bead technology for the development of a high throughput screening method to rapidly measure gene expression changes in allergen-treated DC-like cell lines. The observed effects on gene expression in these cell lines will be compared to data derived from exposing human peripheral blood derived-DC to the same chemical allergens. The goal of this project is to provide a quick, easy and more cost-effective in vitro method to evaluate a large number of chemicals in order to identify genes that when measured in vitro will aid in the prediction of the skin sensitization potential of chemicals in vivo.Development of an in vitro system to test the effect and toxicity of anti-inflammatory and anti-fibrotic drugs in the human liverGeny Groothuis, PhD/Peter OlingaUniversity of GroningenLiver cirrhosis is a fatal liver disease and belongs to the top ten causes of death in the Western World. Cirrhosis is characterized by, among others, excessive deposition of connective tissue (fibrosis) leading to loss of liver function. To date, no adequate drug therapy is available due to the fact that the potential drugs show either lack of effect or serious side effects. The only effective treatment is liver transplantation.

In addition, the pathogenesis of the disease process, being the onset and progression of fibrosis and its reversibility, are only partly understood. Research concerning the pathogenesis and the development of drugs is usually performed in animal experiments, which is accompanied by relatively high discomfort for the animals.

In this project an in vitro system using liver slices will be developed in order to be able to investigate the pathogenesis of the disease without treatment of the animals, thereby reducing the number and the discomfort of the animals to be used. Moreover, the system will allow us to test drugs, developed in our institute for efficacy and toxicity, in vitro instead of in vivo, as is currently the case. In addition, such an in vitro system can be applied to human liver tissue, enabling us to study human specific features of the pathogenesis of liver fibrosis and cirrhosis. This will contribute to a safer first administration of newly developed drugs in man and to a reduction, replacement and refinement of animal experiments.Sea Urchins as an Alternative Animal Model for Testing Drugs and Neurotoxicants as Potential NeuroteratogensJean M. Lauder, Ph.DThe University of North Carolina at Chapel HillThe main goal is to use sea urchin embryos as an alternative animal model to study how exposure to drugs or neurotoxins during pregnancy may cause defects in the developing nervous system. During the first year of funding, a perturb-and-rescue strategy was used to characterize malformations caused by a variety of drugs and neurotoxins, including drugs that target receptors for the brain chemical (neurotransmitter) serotonin, and the neurotoxin, retinoic acid. Results of these studies provided evidence that retinoic acid causes malformations by inhibiting formation of cyclic AMP (cAMP), an important signal regulating cellular development. These malformations can be prevented by drugs that stimulate the cAMP system, like forskolin, or that activate serotonin receptors which promote cAMP synthesis. These results provided the rationale for studies to investigate cellular and molecular mechanisms mediating the opposing actions of retinoic acid and serotonin. The ultimate goal is to use the sea urchin model to test the hypothesis that serotonin opposes retinoid signaling during early stages of development by activating specific receptors and intracellular signaling pathways that promote expression of genes required for the development of specific cell types. Understanding how opposing signaling by retinoic acid and serotonin regulates early sea urchin development could provide important insights into the how the functions of neurotransmitters changed during evolution to allow them to both mediate communication between nerve cells, and act as growth factors for developing cells.Adult Sea UrchinZebrafish as an in vivo model system for identifying developmental neurotoxicantsPamela Lein, PhD and Robert Tanguay, PhDOregon Health & Science University, Oregon State UniversityThere is evidence that exposure of the developing human nervous system to toxic chemicals such as organophosphorus pesticides (OPs) can cause changes in the behavioral, emotional and intellectual function of children. There is, therefore, much interest in screening chemicals to identify those with potential to cause developmental neurotoxicity. Currently approved tests for this purpose primarily use rodents and are complex and expensive in terms of scientific resources, time and animal use. We propose to evaluate the zebrafish as an alternative model for screening chemicals for possible adverse effects on the developing brain. We will use a specific transgenic line of zebrafish (NBT-GFP) in which motor neurons express green fluorescent protein (GFP) to measure axon outgrowth and quantify motor behavior at varying times following exposure to organophosphorus pesticides (OPs). The reasons for choosing the zebrafish include: (1) The cell and molecular mechanisms that control the normal development of the zebrafish nervous system are remarkably similar to those in humans, increasing the validity of using this model to assess potential risks to the developing human nervous system. (2) The expression of green fluorescent protein (GFP) in specific populations of zebrafish neurons coupled with the fact that zebrafish are transparent, allows non-invasive imaging of these GFP-labeled neurons in the same animal over time, thereby significantly reducing the number of animals required for developmental neurotoxicity testing. (3) The small size, rapid nervous system development and short life cycle of zebrafish are favorable for adapting this model system for testing large numbers of chemicals.An in vitro model system to evaluate drug effects on B-lymphocyte survival and antibody productionDonna Muscarella, PhDCornell UniversityThe production of the diverse repertoire of antibodies required to recognize foreign antigens is achieved by the selection of specific clonal populations of B-lymphocytes (B-cells). This fundamental process occurs within the germinal centers of lymph nodes is are dependent on specific survival signals generated within the germinal center. Such signals are derived, in part, by the association/adhesion of B-cells with follicular dendritic cells. Although animal models have been instrumental in identifying potentially immunotoxic chemicals, the basic mechanisms by which chemicals may specifically perturb the germinal center reaction, resulting in aberrant antibody production, are not understood.

Our experimental system is comprised of a panel of human B-cell lines that, when grown in a co-culture system with the human follicular dendritic cell line, HK, specifically adhere to the HK cells. These adhered cells show alterations in their propensity to undergo apoptosis induced by a variety of clinically important drugs and environmental chemicals. Importantly, the B-cell lines show differences in their sensitivity to apoptosis induction by chemicals due to the altered expression of genes encoding the Bcl-2 family of apoptosis-regulating proteins. These genes are also differentially expressed at specific stages of normal B-cell maturation within the germinal center. Thus, this model system will also allow us to identify combinations of normal physiological signals (i.e. adhesion to dendritic cells and expression of Bcl-2 proteins) that may modulate the sensitivity of susceptible and resistant B-cell populations to apoptosis induction by chemical/drug exposure.

We will use this system to examine the effects of potentially immunotoxic chemicals on essential processes in the germinal center reaction including: the interaction of B-cells with HK cells via specific surface receptors, the signaling pathways activated by surface-receptor engagement that control cell survival, and the role of mitochondrial function as a critical target of immunotoxic chemicals. This model system is expected to provide an alternative means to assess and predict the immunotoxic potential of drugs and chemicals on B-cell survival.The impact of housing conditions on the stress response of rats in the laboratory settingMargaret A. RoseProfessor, Faculty of Medicine, UNSW Area Director, Research Management, Prince of Wales Hospital, Randwick NSWObjective: This study will provide new information relevant to the validation of the role of environmental conditions in achieving the goals of Refinement and Reduction. Thus, through simple changes to routine animal husbandry practices, a significant reduction in the level of distress animals may experience under laboratory conditions could be achieved.

Specific Aim: To test whether the physical complexity of the environmental conditions which a rat experiences from weaning affect its ability to cope with potentially stressful experimental procedures.

Background: Providing laboratory animals with conditions which meet their species-specific needs is essential for their wellbeing and various strategies, often described under the general heading of environmental enrichment (EE), have been proposed to achieve this. However, there are important differences in the conditions described as EE in the neurosciences and the ways in which EE is provided for laboratory animals. In the former EE encompasses a complex of environmental experiences designed to enhance the animal's cognitive and emotional development, whereas, the latter most often involves simple modifications to physical structures. However, data from the neurosciences show that the design and complexity of housing conditions influence the behavioural and neurological development of rats with significant effects on cognitive and emotional development.

The evidence that EE affects a rat's capacity to cope with novelty by modulating anxiety and distress, suggests that the construction of the environment has significant implications, not only in achieving comfortable living conditions for laboratory animals but also, in promoting Refinement. To date little attention has been paid to the critical application of these studies to the day to day management of laboratory animal colonies. However, to evaluate the potential benefits of environmental conditions on animal welfare and in achieving Refinement, data relating to emotionality and physiological response to stressors would be informative. These are both areas where there is limited data and the influence of physical environmental enrichment is unclear. The proposed study will focus on both behavioural and neurofunctional measures of emotionality and physiological measures of acute and chronic stress. The influences of physical environmental conditions of varying complexity and responses to psychological and physical stressors will be tested.

Experimental Design: Inbred Wistar rats will be housed in one of three environmental conditions from weaning until the end of the study:

Group 3 — as for Group 2 with PVC tunnels, wooden blocks, and objects (variety of sizes, shapes and colours) which will be changed weekly.

At weaning (D21), pups will be segregated into male and female groups, 4 siblings in each, and will remain in these cohorts for the remainder of the study. On D49, boxes in each environmental condition will be randomly allocated to one of three treatments for the next 7 days —

control

single housing

daily restraint with subcutaneous saline injection

On D57, boxes will be designated either for (1) behavioural tests of emotionality — the open-field test and the elevated maze test will be performed with a 7 day interval between testing - or (2) a functional-anatomical study, involving evoked neuronal activation of brain regions known to mediate emotional behaviours. On D67 (3 days after the second behaviour test), these animals will either undergo an acute restraint test which will measure corticosterone response, or be killed and atria dissected for an in vitro study of beta-adrenoceptor responsiveness.

Relevance to goals of 3R's: Data are relevant to the goals of Refinement and Reduction. Enabling an animal to modulate its response to stressors will promote its wellbeing and reduce the potential of that stress response as a confounding variable in data collection.

Future studies will look at the role of handling, the complimentary influences of handling and EE, the potential benefits of early life experiences and strain differences.Skin Explants for Assessment of Cutaneous Reactions to XenobioticsCraig Svensson, PhDThe University of IowaSkin rashes to chemicals used in a variety of occupational and domestic settings is a common problem that results in discomfort for affected individuals, as well as significant economic costs (estimated to exceed $200 million annually). Currently, new chemical compounds are tested in animals to predict their ability to cause skin rashes in humans. These animal tests are expensive, time consuming, and possess limited predictive capacity. The objective of this project is to develop the means to use waste human skin from surgical procedures as an alternative to animal tests to predict the ability of chemicals to cause skin rashes. Studies have demonstrated that rash-producing chemicals placed on the skin cause the migration of a specific type of cell (dendritic cells) from the skin. These cells are critical in the initiation of an immune response that results in the skin rash. Our project will determine the ability of compounds known to cause skin rashes to provoke the migration of dendritic cells in a human skin explant model. The human skin explant represents small pieces of skin from patients undergoing surgery that requires a reduction in skin. These explants can be maintained for up to 72 hours. We will assess dendritic cell migration from the explants after the application of various chemicals. If successful in demonstrating the ability of such compounds to provoke the migration of dendritic cells, we will then develop the means by which this assessment can be made using an automated system and very small samples.Morphologic and genomic approaches to evaluate the zebrafish embryo test as an alternative in vitro screening model system to predict developmental toxicityHilda Witters, PhDVITO - Flemish Institute for Technological ResearchDevelopmental toxicity is a major issue in children's health worldwide. The developing human system is susceptible to many toxicants, and chemical exposure during development may cause lasting metabolic deficits. Such damage can range from subtle to severe, and it may impose substantial burdens on affected individuals, their families, and society. Testing compounds for developmental toxicity endpoints is an important societal and scientific goal.

Zebrafish is an established animal model for many aspects of human development and diseases that will benefit from extended embryological knowledge and genomic research. The first specific aim of the project will be the determination of the developmental time course of early physical malformations and neurobehavioural dysfunctions caused by a selection of candidate compounds in a dose range matter. The second aim will be a detailed investigation of the cellular and molecular mechanisms (fingerprints of genes), contributing to these (d)effects as observed for the most potent compounds.

The overall purpose of this research project is to utilize zebrafish embryos, as a simple and fast high-throughput assay to predict for potential malformations and dysfunctional effects during early development, caused by exposure to different classes of chemicals. This short term zebrafish assay might in the first place replace and reduce the number of mammals currently used for testing of chemicals. And eventually as it does contribute to an improved and cost-efficient test strategy for numerous chemicals, it may lead to reduction or prevention of the majority of induced birth defects.